The present invention related to the field of Alzheimer""s Disease. APP, amyloid beta peptide, and human aspartyl proteases as well as a method for the identification of agents that modulate the activity of these polypeptides.
Alzheimer""s disease (AD) causes progressive dementia with consequent formation of amyloid plaques, neurofibrillary tangles, gliosis and neuronal loss. The disease occurs in both genetic and sporadic forms whose clinical course and pathological features are quite similar. Three genes have been discovered to date which when mutated cause an autosomal dominant form of Alzheimer""s disease. These encode the amyloid protein precursor (APP) and two related proteins, presenilin-1 (PS1) and presenilin-2 (PS2), which as their names suggest are both structurally and functionally related. Mutations in any of the three enhance proteolytic processing of APP via an intracellular pathway that produces amyloid beta peptide or the Axcex2 peptide (or sometimes here as Abeta), a 40-42 amino acid long peptide that is the primary component of amyloid plaque in AD. Dysregulation of intracellular pathways for proteolytic processing may be central to the pathophysiology of AD. In the case of plaque formation, mutations in APP, PS1 or PS2 consistently alter the proteolytic processing of APP so as to enhance formation of Axcex2 1-42, a form of the Axcex2 peptide which seems to be particularly amyloidogenic, and thus very important in AD. Different forms of APP range in size from 695-770 amino acids, localize to the cell surface, and have a single C-terminal transmembrane domain. The Abeta peptide is derived from a region of APP adjacent to and containing a portion of the transmembrane domain. Normally, processing of APP at the xcex1-secretase site cleaves the midregion of the Axcex2 sequence adjacent to the membrane and releases the soluble, extracellular domain of APP from the cell surface. This xcex1-secretase APP processing, creates soluble APP-xcex1, and it is normal and not thought to contribute to AD.
Pathological processing of APP at the xcex2- and xcex3-secretase sites produces a very different result than procesing at the xcex1 site. Sequential processing at the xcex2- and xcex3-secretase sites releases the Axcex2 peptide, a peptide possibly very important in AD pathogenesis. Processing at the xcex2- and xcex3-secretase sites can occur in both the endoplasmic reticulum (in neurons) and in the endosomal/lysosomal pathway after reinternalization of cell surface APP (in all cells). Despite intense efforts, for 10 years or more, to identify the enzymes responsible for processing APP at the xcex2 and xcex3 sites, to produce the xcex2 peptide, those proteases remained unknown until this disclosure. Here, for the first time, we report the identification and characterization of the xcex2 secretase enzyme. We disclose some known and some novel human aspartic proteases that can act as xcex2-secretase proteases and, for the first time, we explain the role these proteases have in AD. We describe regions in the proteases critical for their unique function and for the first time characterize their substrate. This is the first description of expressed isolated purified active protein of this type, assays that use the protein, in addition to the identification and creation of useful cell lines and inhibitors.
Here we disclose a number of variants of the asp2 gene and peptide.
The invention provides for an isolated or purified nucleic acid polynucleotide that codes for a protease capable of cleaving the beta (xcex2) secretase cleavage site of APP that contains two or more sets of special nucleic acids, where the special nucleic acids are separated by nucleic acids that code for about 100 to 300 amino acid positions, where the amino acids in those positions may be any amino acids, where the first set of special nucleic acids consists of the nucleic acids that code for the peptide DTG, where the first nucleic acid of the first special set of nucleic acids is, the first special nucleic acid, and where the second set of nucleic acids code for either the peptide DSG or DTG, where the last nucleic acid of the second set of nucleic acids is the last special nucleic acid, with the proviso that the nucleic acids disclosed in SEQ ID NO. 1 and SEQ. ID NO. 5 are not included. In one embodiment, the two sets of nucleic acids are separated by nucleic acids that code for about 125 to 222 amino acid positions, which may be any amino acids. In a particular embodiment, the two sets of special nucleic acids are separated by nucleic acids that code for about 150 to 172 amino acid positions, which may be any amino acids. In a more particular embodiment, the two sets are separated by nucleic acids that code for about 172 amino acid positions, which may be any amino acids. An exemplary polynucleotide comprises the nucleic acid described in SEQ. ID. NO. 3. In another embodiment, the two sets of nucleic acids are separated by nucleic acids that code for about 150 to 196 amino acid positions. In another embodiment, the two sets of nucleotides are separated by nucleic acids that code for about 196 amino acids. An exemplary polynucleotide comprises the two sets of nucleic acids separated by the same nucleic acid sequences that separate the same set of special nucleic acids in SEQ. ID. NO. 5. In a particular embodiment, the two sets of nucleic acids are separated by nucleic acids that code for about 150 to 190, amino acid. In another embodiment, the two sets of nucleotides are separated by nucleic acids that code for about 190 amino acids. In a more particular embodiment, the two sets of nucleotides are separated by the same nucleic acid sequences that separate the same set of special nucleotides in SEQ. ID. NO. 1. In one embodiment, the first nucleic acid of the first special set of amino acids, that is, the first special nucleic acid, is operably linked to any codon where the nucleic acids of that codon codes for any peptide comprising from 1 to 10,000 amino acid. In one variation, the first special nucleic acid is operably linked to nucleic acid polymers that code for any peptide selected from the group consisting of: any any reporter proteins or proteins which facilitate purification. In another variation, the first special nucleic acid is operably linked to nucleic acid polymers that code for any peptide selected from the group consisting of: immunoglobin-heavy chain, maltose binding protein, glutathion S transfection, Green Fluorescent protein, and ubiquitin. In another embodiment, the last nucleic acid of the second set of special amino acids, that is, the last special nucleic acid, is operably linked to nucleic acid polymers that code for any peptide comprising any amino acids from 1 to 10,000 amino acids. In one variation, the last special nucleic acid is operably linked to any codon linked to nucleic acid polymers that code for any peptide selected from the group consisting of: any reporter proteins or proteins which facilitate purification. In another embodiment, the first special nucleic acid is operably linked to nucleic acid polymers that code for any peptide selected from the group consisting of: immunoglobin-heavy chain, maltose binding protein, glutathion S transfection, Green Fluorescent protein, and ubiquitin.
The invention provides for an isolated or purified nucleic acid polynucleotide that codes for a protease capable of cleaving the beta secretase cleavage site of APP that contains two or more sets of special nucleic acids, where the special nucleic acids are separated by nucleic acids that code for about 100 to 300 amino acid positions, where the amino acids in those positions may be any amino acids, where the first set of special nucleic acids consists of the nucleic acids that code for DTG, where the first nucleic acid of the first special set of nucleic acids is, the first special nucleic acid, and where the second set of nucleic acids code for either DSG or DTG, where the last nucleic acid of the second set of special nucleic acids is the last special nucleic acid, where the first special nucleic acid is operably linked to nucleic acids that code for any number of amino acids from zero to 81 amino acids and where each of those codons may code for any amino acid. In an embodiment, the first special nucleic acid is operably linked to nucleic acids that code for any number of from 64 to 77 amino acids where each codon may code for any amino acid. In a particular embodiment, the first special nucleic acid is operably linked to nucleic acids that code for 71 amino acids. For example, the first special nucleic acid is operably linked to 71 amino acids and where the first of those 71 amino acids is the amino acid T. In another embodiment, the polynucleotide comprises a sequence that is at least 95% identical to a human Asp1 or Asp2 sequence as taught herein. In still another embodiment, the first special nucleic acid is operably linked to nucleic acids that code for any number of from 40 to 54 amino acids where each codon may code for any amino acid. In a particular embodiment, the first special nucleic acid is operably linked to nucleic acids that code for 47 amino acids. For example, the first special nucleic acid is operably linked to 47 codons where the first those 47 amino acids is the amino acid E such as a polynucleotide comprising a sequence that is at least 95% identical to SEQ. ID. NO: 29 described in Example 10 or the complete polynucleotide sequence of SEQ. ID. NO: 29 described in Example 10.
In another related aspect, the invention provides for an isolated or purified nucleic acid polynucleotide that codes for a protease capable of cleaving the beta (xcex2) secretase cleavage site of APP that contains two or more sets of special nucleic acids, where the special nucleic acids are separated by nucleic acids that code for about 100 to 300 amino acid positions, where the amino acids in those positions may be any amino acids, where the first set of special nucleic acids consists of the nucleic acids that code for the peptide DTG, where the first nucleic acid of the first special set of amino acids is, the first special nucleic acid, and where the second set of special nucleic acids code for either the peptide DSG or DTG, where the last nucleic acid of the second set of special nucleic acids, the last special nucleic acid, is operably linked to nucleic acids that code for any number of codons from 50 to 170 codons. In an embodiment, the last special nucleic acid is operably linked to nucleic acids comprising from 100 to 170 codons. In a particular embodiment, the last special nucleic acid is operably linked to nucleic acids comprising from 142 to 163 codons. In another embodiment, the last special nucleic acid is operably linked to nucleic acids comprising about 142 codons. For example, the polynucleotide comprises a sequence that is at least 95% identical to SEQ. ID NO: 21 described in Example 9 or SEQ ID NO: 29 described in Example 10 or the complete polynucleotide sequence of SEQ. ID. NO: 21 described in Example 9 or SEQ ID NO: 29 described in Example 10. In one variation, the last special nucleic acid is operably linked to nucleic acids comprising about 163 codons. In another variation, the last special nucleic acid is operably linked to nucleic acids comprising about 170 codons. In another embodiment, the second set of special nucleic acids code for the peptide DSG, and optionally the first set of nucleic acid polynucleotide is operably linked to a peptide purification tag. For example, the nucleic acid polynucleotide is operably linked to a peptide purification tag which is six histidine. In still another embodiment, the first set of special nucleic acids are on one polynucleotide and the second set of special nucleic acids are on a second polynucleotide, where both first and second polynucleotides have at least 50 codons. In one embodiment of this type, the first set of special nucleic acids are on one polynucleotide and the second set of special nucleic acids are on a second polynucleotide, where both first and second polynucleotides have at least 50 codons where both said polynucleotides are in the same solution. In a related aspect, the invention provides for a vector which contains a polynucleotide as described above, and a cell or cell line which contains a polynucleotide described above.
In still another aspect, the invention provides an isolated or purified peptide or protein comprising an amino acid polymer that is a protease capable of cleaving the beta (xcex2) secretase cleavage site of APP that contains two or more sets of special amino acids, where the special amino acids are separated by about 100 to 300 amino acid positions, where each amino acid position can be any amino acid, where the first set of special amino acids consists of the peptide DTG, where the first amino acid of the first special set of amino acids is, the first special amino acid, where the second set of amino acids is selected from the peptide comprising either DSG or DTG, where the last amino acid of the second set of special amino acids is the last special amino acid, with the proviso that the proteases disclosed in SEQ ID NO. 2 and SEQ ID NO. 6 are not included. In an embodiments, the two sets of amino acids are separated by about 125 to 222 amino acid positions where in each position it may be any amino acid. In a particular embodiment, the two sets of amino acids are separated by about 150 to 172 amino acids. In another particular embodiment, the two sets of amino acids are separated by about 172 amino acids. For example, the polypeptide is a the protease described in SEQ. ID. NO. 4. In another embodiment, the two sets of amino acids are separated by about 150 to 196 amino acids. In one variation, the two sets of amino acids are separated by about 196 amino acids. In an embodiment, the two sets of amino acids are separated by the same amino acid sequences that separate the same set of special amino acids in SEQ. ID. NO. 6. In a particular embodiment, the two sets of amino acids are separated by about 150 to 190, amino acids. In another particular embodiment, the two sets of nucleotides are separated by about 190 amino acids. For example, the two sets of nucleotides are separated by the same amino acid sequences that separate the same set of special amino acids in SEQ. ID. NO. 2. In one embodiment, the first amino acid of the first special set of amino acids, that is, the first special amino acid, is operably linked to any peptide comprising from 1 to 10,000 amino acids. In another embodiment, the first special amino acid is operably linked to any peptide selected from the group consisting of: any any reporter proteins or proteins which facilitate purification. In particular embodiments, the first special amino acid is operably linked to any peptide selected from the group consisting of: immunoglobin-heavy chain, maltose binding protein, glutathion S transfection, Green Fluorescent protein, and ubiquitin. In still another variation, the last amino acid of the second set of special amino acids, that is, the last special amino acid, is operably linked to any peptide comprising any amino acids from 1 to 10,000 amino acids. By way of nonlimiting example, the last special amino acid is operably linked any peptide selected from the group consisting of any reporter proteins or proteins which facilitate purification. In particular embodiments, the first special amino acid is operably linked to any peptide selected from the group consisting of: immunoglobin-heavy chain, maltose binding protein, glutathion S transfection, Green Fluorescent protein, and ubiquitin.
The invention also provides for an isolated or purified peptide or protein comprising an amino acid polypeptide that codes for a protease capable of cleaving the beta secretase cleavage site of APP that contains two or more sets of special amino acids, where the special amino acids are separated by about 100 to 300 amino acid positions, where each amino acid in each position can be any amino acid, where the first set of special amino acids consists of the amino acids DTG, where the first amino acid of the first special set of amino acids is, the first special amino acid, D, and where the second set of amino acids is either DSG or DTG, where the last amino acid of the second set of special amino acids is the last special amino acid, G, where the first special amino acid is operably linked to amino acids that code for any number of amino acids from zero to 81 amino acid positions where in each position it may be any amino acid. In one embodiment, the first special amino acid is operably linked to a peptide from about 64 to 77 amino acids positions where each amino acid position may be any amino acid. In a particular embodiment, the first special amino acid is operably linked to a peptide of 71 amino acids. In a more particular embodiment, the first special amino acid is operably linked to 71 amino acids and the first of those 71 amino acids is the amino acid T. For example, the polypeptide comprises a sequence that is at least 95% identical to an aspartyl protease sequence as described herein. In another embodiment, the first special amino acid is operably linked to any number of from 40 to 54 amino acids where each amino acid position may be any amino acid. In a particular embodiment, the first special amino acid is operably linked to amino acids that code for a peptide of 47 amino acids. In a very particular embodiment, the first special amino acid is operably linked to a 47 amino acid peptide where the first those 47 amino acids is the amino acid E. For example, the polypeptide comprises a sequence that is at least 95% identical to SEQ. ID. NO: 30 described in Example 10 or the complete polypeptide sequence of SEQ ID NO: 30 described in Example 10.
In still another related aspect, an isolated or purified amino acid polypeptide that is a protease capable of cleaving the beta (xcex2) secretase cleavage site of APP that contains two or more sets of special amino acids, where the special amino acids are separated by about 100 to 300 amino acid positions, where each amino acid in each position can be any amino acid, where the first set of special amino acids consists of the amino acids that code for DTG, where the first amino acid of the first special set of amino acids is, the first special amino acid, D, and where the second set of amino acids are either DSG or DTG, where the last amino acid of the second set of special amino acids is the last special amino acid, G, which is operably linked to any number of amino acids from 50 to 170 amino acids, which may be any amino acids. In one embodiment, the last special amino acid is operably linked to a peptide of about 100 to 170 amino acids. In a particular embodiment, the last special amino acid is operably linked to to a peptide of about 142 to 163 amino acids. In another particular embodiment, the last special amino acid is operably linked to to a peptide of about about 142 amino acids. For example, the polypeptide comprises a sequence that is at least 95% identical to SEQ. ID. NO: 22 described in Example 9 or SEQ ID NO: 30 described in Example 10. In one particular embodiment, the last special amino acid is operably linked to a peptide of about 163 amino acids. For example, the polypeptide comprises a sequence that is at least 95% identical to SEQ. ID. NO: 22 described in Example 9 or SEQ ID NO: 30 described in Example 10, or the complete polypeptide sequence of SEQ. ID. NO: 22 described in Example 9 or SEQ ID NO: 30 described in Example 10. The amino acid polypeptide of claim 74 where In another embodiment, the last special amino acid is operably linked to to a peptide of about 170 amino acids. In a particular embodiment, the second set of special amino acids is comprised of the peptide with the amino acid sequence DSG. Optionally, the amino acid polypeptide is operably linked to a peptide purification tag such as a peptide purification tag which is six histidine. In one variation, the first set of special amino acids are on one polypeptide and the second set of special amino acids are on a second polypeptide, where both first and second polypeptide have at lease 50 amino acids, which may be any amino acids. In another variation, the first set of special amino acids are on one polypeptide and the second set of special amino acids are on a second polypeptide, where both first and second polypeptides have at lease 50 amino acids where both said polypeptides are in the same vessel. The invention also provides for a vector which contains a polypeptide as described herein. The invention further provides for a cell or cell line which contains a polynucleotide described herein. The invention also provides for a process of making any of the polynucleotides, vectors, or cells described herein, and a process of making any of the polypeptides described herein.
Any isolated or purified peptide or protein comprising an amino acid polypeptide that codes for a protease capable of cleaving the beta secretase cleavage site of APP that contains two or more sets of special amino acids, where the special amino acids are separated by about 100 to 300 amino acid positions, where each amino acid in each position can be any amino acid, where the first set of special amino acids consists of the amino acids DTG, where the first amino acid of the first special set of amino acids is, the first special amino acid, D, and where the second set of amino acids is either DSG or DTG, where the last amino acid of the second set of special amino acids is the last special amino acid. G, where the first special amino acid is operably linked to amino acids that code for any number of amino acids from zero to 81 amino acid positions where in each position it may be any amino acid.
The invention provides for an amino acid polypeptide described herein, where the first special amino acid is operably linked to a peptide from about 30 to 77 amino acids positions where each amino acid position may be any amino acid. The invention also provides for an amino acid polypeptide described herein, where the first special amino acid is operably linked to a peptide of 35, 47, 71, or 77 amino acids.
The invention provides for an amino acid polypeptide described herein, where the first special amino acid is operably linked to the same corresponding peptides from SEQ. ID. NO. 3 that are 35, 47, 71, or 77 peptides in length, beginning counting with the amino acids on the first special sequence, DTG, towards the N-terminal of SEQ. ID. NO. 3.
The invention provides for an amino acid polypeptide described herein, where the polypeptide comprises a sequence that is at least 95% identical to the same corresponding amino acids in SEQ. ID. NO. 4, that is, identical to that portion of the sequences in SEQ. ID. NO. 4, including all the sequences from both the first and or the second special nucleic acids, toward the N-terminal, through and including 71, 47, 35 amino acids before the first special amino acids. (Examples 10 and 11).
The invention provides for an amino acid polypeptide described herein, where the complete polypeptide comprises the peptide of 71 amino acids, where the first of the amino acid is T and the second is Q. The invention also provides for a nucleic acid polynucleotide described herein, where the polynucleotide comprises a sequence that is at least 95% identical to the same corresponding amino acids in SEQ. ID. NO. 3, that is, identical to the sequences in SEQ. ID. NO. 3 including the sequences from both the first and or the second special nucleic acids, toward the N-Terminal, through and including 71 amino acids, see Example 10, beginning from the DTG site and including the nucleotides from that code for 71 amino acids).
The invention provides for a nucleic acid polynucleotide described herein, where the complete polynucleotide comprises identical to the same corresponding amino acids in SEQ. ID. NO. 3, that is, identical to the sequences in SEQ. ID. NO. 3 including the sequences from both the first and or the second special nucleic acids, toward the N-Terminal, through and including 71 amino acids, see Example 10, beginning from the DTG site and including the nucleotides from that code for 71 amino acids).
The invention provides for a nucleic acid polynucleotide described herein, where the first special nucleic acid is operably linked to nucleic acids that code for any number of from about 30 to 54 amino acids where each codon may code for any amino acid.
The invention provides for a nucleic acid polynucleotide described herein, where the first special nucleic acid is operably linked to 47 codons where the first those 35 or 47 amino acids is the amino acid E or G.
The invention provides for a nucleic acid polynucleotide described herein, where the polynucleotide comprises a sequence that is at least 95% identical to the same corresponding amino acids in SEQ. ID. NO. 3, that is, identical to that portion of the sequences in SEQ. ID. NO. 3 including the sequences from both the first and or the second special nucleic acids, toward the N-Terminal, through and including 35 or 47 amino acids, see Example 11 for the 47 example, beginning from the DTG site and including the nucleotides from that code for the previous 35 or 47 amino acids before the DTG site The nucleic acid polynucleotide of the present invention, where the polynucleotide comprises identical to the same corresponding amino acids in SEQ. ID. NO. 3, that is, identical to the sequences in SEQ. ID. NO. 3 including the sequences from both the first and or the second special nucleic acids, toward the N-Terminal, through and including 35 or 47 amino acids, see Example 11 for the 47 example, beginning from the DTG site and including the nucleotides from that code for the previous 35 or 47 amino acids before the DTG site.
An isolated nucleic acid molecule comprising a polynucleotide, said polynucleotide encoding a Hu-Asp polypeptide and having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of:
(a) a nucleotide sequence encoding a Hu-Asp polypeptide selected from the group consisting of Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b), wherein said Hu-Asp1, Hu-Asp2(a) and Hu-Asp2(b) polypeptides have the complete amino acid sequence of SEQ ID No. 2, SEQ ID No. 4, and SEQ ID No. 6, respectively; and
(b) a nucleotide sequence complementary to the nucleotide sequence of (a).
The invention provide for a nucleic acid molecule, wherein said Hu-Asp polypeptide is Hu-Asp1, and said polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO:1; and a nucleic acid molecule, wherein said Hu-Asp polypeptide is Hu-Asp2(a), and said polynucleotide molecule comprises the nucleotide sequence of SEQ ID NO:3; and a nucleic acid molecule that encodes a Hu-Asp polypeptide that is Hu-Asp2(b), and said polynucleotide molecule comprises the nucleotide sequence of SEQ ID No. 5. The invention provides for an isolated nucleic acid molecule comprising polynucleotide which hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence described above. The invention also provides for a vector comprising the nucleic acid molecule described herein. Optionally, the vector contains a nucleic acid molecule operably linked to a promoter for the expression of a Hu-Asp polypeptide such as Hu-Asp1, Hu-Asp2(a) or Hu-Asp2(b). The invention provides for a host cell comprising the vector described above. The invention also provides for a method of obtaining a Hu-Asp polypeptide comprising culturing the host cell described above and isolating said Hu-Asp polypeptide. The invention provides for an isolated Hu-Asp1 polypeptide comprising an amino acid sequence at least 95% identical to a sequence comprising the amino acid sequence of SEQ ID No. 2, an isolated Hu-Asp2(a) polypeptide comprising an amino acid sequence at least 95% identical to a sequence comprising the amino acid sequence of SEQ ID No. 4, and an isolated Hu-Asp2(a) polypeptide comprising an amino acid sequence at least 95% identical to a sequence comprising the amino acid sequence of SEQ ID No. 8. The invention also provides for an isolated antibody that binds specifically to the Hu-Asp polypeptide described herein.
Here we disclose numerous methods to assay the enzyme.
The invention provides for a method to identify a cell that can be used to screen for inhibitors of xcex2 secretase activity comprising:
(a) identifying a cell that expresses a protease capable of cleaving APP at the xcex2 secretase site, comprising:
i) collect the cells or the supernatant from the cells to be identified
ii) measure the production of a critical peptide, where the critical peptide is selected from the group consisting of either the APP C-terminal peptide or soluble APP,
iii) select the cells which produce the critical peptide.
In one embodiment, the cells are collected and the critical peptide is the APP C-terminal peptide created as a result of the xcex2 secretase cleavage. In another embodiment, the supernantent is collected and the critical peptide is soluble APP where the soluble APP has a C-terminal created by xcex2 secretase cleavage. In one variation, the cells contain any of the nucleic acids or polypeptides described above and where the cells are shown to cleave the xcex2 secretase site of any peptide having the following peptide structure, P2, P1, P1xe2x80x2, P2xe2x80x2, where P2 is K or N, where P1 is M or L, where P1xe2x80x2 is D, where P2xe2x80x2 is A. In another vatiation P2 is K and P1 is M or P2 is N and P1 is L.
The invention provides for a bacterial cell comprising any nucleic acids or peptides described above. For example, bacterial cell that is E coli. The invention also provides for an eukaryotic cell comprising any nucleic acids or polypeptides described above.
The invention provides for an insect cell comprising any of the nucleic acids or polypeptides described above. These insect cells contemplated include sf9, and High 5. The invention also provides for a mammalian cell comprising any of the nucleic acids or polypeptides described herein. An exemplary mammalian cell may be selected from the group consisting of, human, rodent, lagomorph, and primate. An exemplary human cell may be selected from the group comprising HEK293, and IMR-32. An exemplary primate cell may be a COS-7 cell. A rodent cell may be selected from, CHO-K1, Neuro-2A, and 3T3 cells. The invention also provides for a yeast cell or an avian cell comprising any of the nucleic acids or polypeptides described above.
The invention provides for an isoform of APP where the last two carboxy terminus amino acids of that isoform are both lysine residues. An isoform is any APP polypeptide, including APP variants (including mutations), and APP fragments that exists in humans such as those described in U.S. Pat. No. 5,766,846, col 7, lines 45-67, incorporated into this document by reference. One embodiment is an isoform of APP, comprising the isoform known as APP695 modified so that its last two having two lysine residues as its last two carboxy terminus amino acids. For example, an APP isoform comprising SEQ ID NO:16, or the APP isoform variant comprising SEQ. ID. NO. 18, or 20. The invention also provides for an eukaryotic cell line, comprising nucleic acids encoding modified APP isoforms or polypeptides comprising modified APP isoforms. The cell line may be a mammalian cell line (HEK293, Neuro2a). The invention also provides for a method for identifying inhibitors of an enzyme that cleaves the beta secretase cleavable site of APP comprising:
a) culturing cells in a culture medium under conditions in which the enzyme causes processing of APP and release of amyloid beta-peptide into the medium and causes the accumulation of CTF99 fragments of APP in cell lysates.
b) exposing the cultured cells to a test compound; and specifically determining whether the test compound inhibits the function of the enzyme by measuring the amount of amyloid beta-peptide released into the medium and or the amount of CTF99 fragments of APP in cell lysates;
c) identifying test compounds diminishing the amount of soluble amyloid beta peptide present in the culture medium and diminution of CTF99 fragments of APP in cell lysates as Asp2 inhibitors.
In an exemplary embodiments, the cultured cells are a human, rodent or insect cell line. It is also contemplated that the human or rodent cell line exhibits xcex2 secretase activity in which processing of APP occurs with release of amyloid beta-peptide into the culture medium and accumulation of CTF99 in cell lysates. Among the contemplated test compounds are antisense oligomers directed against the enzyme that exhibits xcex2 secretase activity, reduces release of soluble amyloid beta-peptide into the culture medium and accumulation of CTF99 in cell lysates. A method for the identification of an agent that decreases the activity of a Hu-Asp polypeptide selected from the group consisting of Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b), the method comprising:
a) determining the activity of said Hu-Asp polypeptide in the presence of a test agent and in the absence of a test agent; and
b) comparing the activity of said Hu-Asp polypeptide determined in the presence of said test agent to the activity of said Hu-Asp polypeptide determined in the absence of said test agent;
whereby a lower level of activity in the presence of said test agent than in the absence of said test agent indicates that said test agent has decreased the activity of said Hu-Asp polypeptide. The nucleic acids, peptides, proteins, vectors, cells and cell lines, and assays described herein.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide that codes for a polypeptide selected from the group consisting of human aspartyl proteases. In particular, human aspartyl protease 1 (Hu-Asp1) and two alternative splice variants of human aspartyl protease 2 (Hu-Asp2), designated herein as Hu-Asp2(a) and Hu-Asp2(b). As used herein, all references to xe2x80x9cHu-Aspxe2x80x9d should be understood to refer to all of Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b). In addition, as used herein, all references to xe2x80x9cHu-Asp2xe2x80x9d should be understood to refer to both Hu-Asp2(a) and Hu-Asp2(b). Hu-Asp1 is expressed most abundantly in pancreas and prostate tissues, while Hu-Asp2(a) and Hu-Asp2(b) are expressed most abundantly in pancreas and brain tissues. The invention also provides isolated Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b) polypeptides, as well as fragments thereof which exhibit aspartyl protease activity.
In a preferred embodiment, the nucleic acid molecules comprise a polynucleotide having a nucleotide sequence selected from the group consisting of residues 1-1554 of SEQ ID NO:1, encoding Hu-Asp1, residues 1-1503 of SEQ ID NO:3, encoding Hu-Asp2(a), and residues 1-1428 of SEQ ID NO:5, encoding Hu-Asp2(b). In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide encoding Hu-Asp1, Hu-Asp2(a), Hu-Asp-2(b), or fragments thereof. European patent application EP 0 848 062 discloses a polypeptide referred to as xe2x80x9cAsp 1,xe2x80x9d that bears substantial homology to Hu-Asp1, while international application WO 98/22597 discloses a polypeptide referred to as xe2x80x9cAsp 2,xe2x80x9d that bears substantial homology to Hu-Asp2(a).
The present invention also provides vectors comprising the isolated nucleic acid molecules of the invention, host cells into which such vectors have been introduced, and recombinant methods of obtaining a Hu-Asp1, Hu-Asp2(a), or Hu-Asp2(b) polypeptide comprising culturing the above-described host cell and isolating the relevant polypeptide.
In another aspect, the invention provides isolated Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b) polypeptides, as well as fragments thereof. In a preferred embodiment, the Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b) polypeptides have the amino acid sequence given in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, respectively. The present invention also describes active forms of Hu-Asp2, methods for preparing such active forms, methods for preparing soluble forms, methods for measuring Hu-Asp2 activity, and substrate for Hu-Asp2 cleavage. The invention also describes antisense oligomers targeting the Hu-Asp1, Hu-Asp2(a) and Hu-Asp2(b) mRNA transcripts and the use of such antisense reagents to decrease such mRNA and consequently the production of the corresponding polypeptide. Isolated antibodies, both polyclonal and monoclonal, that binds specifically to any of the Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b) polypeptides of the invention are also provided.
The invention also provides a method for the identification of an agent that modulates the activity of any of Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b). The inventions describes methods to test such agents in cell-free assays to which Hu-Asp2 polypeptide is added, as well as methods to test such agents in human or other mammalian cells in which Hu-Asp2 is present.
Sequence ID No. 1xe2x80x94Human Asp-1, nucleotide sequence
Sequence ID No. 2xe2x80x94Human Asp-1, predicted amino acid sequence
Sequence ID No. 3xe2x80x94Human Asp-2(a), nucleotide sequence
Sequence ID No. 4xe2x80x94Human Asp-2(a), predicted amino acid sequence
Sequence ID No. 5xe2x80x94Human Asp-2(b), nucleotide sequence
Sequence ID No. 6xe2x80x94Human Asp-2(b), predicted amino acid sequence
Sequence ID No. 7xe2x80x94Murine Asp-2(a), nucleotide sequence
Sequence ID No. 8xe2x80x94Murine Asp-2(a), predicted amino acid sequence
Sequence ID No. 9xe2x80x94Human APP695, nucleotide sequence
Sequence ID No.10xe2x80x94Human APP695, predicted amino acid sequence
Sequence ID No.11xe2x80x94Human APP695-Sw, nucleotide sequence
Sequence ID No.12xe2x80x94Human APP695-Sw, predicted amino acid sequence
Sequence ID No.13xe2x80x94Human APP695-VF, nucleotide sequence
Sequence ID No.14xe2x80x94Human APP695-VF, predicted amino acid sequence
Sequence ID No.15xe2x80x94Human APP695-KK, nucleotide sequence
Sequence ID No.16xe2x80x94Human APP695-KK, predicted amino acid sequence
Sequence ID No.17xe2x80x94Human APP695-Sw-KK, nucleotide sequence
Sequence ID No.18xe2x80x94Human APP695-Sw-KK, predicted amino acid sequence
Sequence ID No.19xe2x80x94Human APP695-VF-KK, nucleotide sequence
Sequence ID No.20xe2x80x94Human APP695-VF-KK, predicted amino acid sequence
Sequence ID No.21xe2x80x94T7-Human-pro-Asp-2(a)xcex94TM, nucleotide sequence
Sequence ID No.22xe2x80x94T7-Human-pro-Asp-2(a)xcex94TM, amino acid sequence
Sequence ID No.23xe2x80x94T7-Caspase-Human-pro-Asp-2(a)xcex94TM, nucleotide sequence
Sequence ID No.24xe2x80x94T7-Caspase-Human-pro-Asp-2(a)xcex94TM, amino acid sequence
Sequence ID No.25xe2x80x94Human-pro-Asp-2(a)xcex94TM (low GC), nucleotide sequence
Sequence ID No.26xe2x80x94Human-pro-Asp-2(a)xcex94TM, (low GC), amino acid sequence
Sequence ID No.27xe2x80x94T7-Caspase-Caspase 8 cleavage-Human-pro-Asp-2(a)xcex94TM, nucleotide sequence
Sequence ID No.28xe2x80x94T7-Caspase-Caspase 8 cleavage-Human-pro-Asp-2(a)xcex94TM, amino acid sequence
Sequence ID No.29xe2x80x94Human Asp-2(a)xcex94TM, nucleotide sequence
Sequence ID No.30xe2x80x94Human Asp-2(a)xcex94TM, amino acid sequence
Sequence ID No.31xe2x80x94Human Asp-2(a)xcex94TM(His)6, nucleotide sequence
Sequence ID No.32xe2x80x94Human Asp-2(a)xcex94TM(His)6, amino acid sequence
Sequence ID No.s 33-46 are described below in the Detailed Description of the Invention.